Skeletal fractures of the long bones are a major orthopedic problem. Skeletal healing is impaired in 10- 20% of these fractures, resulting in nonunion or delayed union healing. This constitutes a highly significant morbidity to patients, prolonged hospitalizations, and a huge economic burden for the patient and society. Thus, it is highly desirable to have an effective treatment to promote bone healing in conditions where the injury is so severe that normal healing is not possible, or in otherwise normal fracture healing impaired by health-related conditions. A robust periosteal response is critical for efficient bone healing. We have developed significant preliminary evidence that ephrin (efn) B1 signaling regulates bone formation and promotes a substantial periosteal response to mechanical loading and fracture repair. Accordingly, we have proposed that efnB1 promotes the periosteal response to loading and injury, and that this periosteal response can be used to enhance the healing of segmental defect injuries. Our laboratory has also recently developed a novel model of segmental bone injury in the mouse femur that is a better paradigm of endochondral bone repair and is more clinically relevant to impaired fracture healing than the commonly used closed fracture models or rigid external fixation approaches to segmental defects. This defect model fails to heal well after it should have achieved bony union, and therefore represents a very challenging model of bone repair to investigate efnB1 regulation of bone healing. Three specific objectives are proposed to test efnB1 regulation of bone repair using this model. 1) To test the hypothesis that efnB1 regulates bone repair in a femoral segmental defect model of bone healing, we will characterize impaired segmental defect healing in a transgenic efnB1 mouse line that overexpresses efnB1 specifically in osteoblast lineage cells. 2) To test the hypothesis that efnB1 enhancement of healing in a segmental defect model of bone repair is regulated by efnB1 reverse signaling that is induced upon efnB1 contact with its receptors (eph), we will examine segmental defect repair in osteoblast-specific efnB1 knockout mice, but selectively restore forward and reverse signaling or only forward signaling by the transduction of appropriate efnB1 gene constructs. These constructs will be either a full-length wild-type efnB1 capable of forward and reverse signaling, or a truncated efnB1 with a defective signaling domain that eliminates reverse signaling. 3) To test the hypothesis that efnB1 therapy can promote the healing of non-union femoral segmental defects, we will utilize the signaling regulation identified in the second specific objective to aply efnB1 and ephB2 gene therapy to the femoral segmental defect of wild-type mice and evaluate healing. Our proposed studies will improve our understanding of ephrin regulation of bone repair in a severely impaired model of healing and evaluate efnB1 therapy as an approach for the improvement of bone healing impaired by clinically challenging injuries, as well by physiological conditions such as osteoporosis.